Project 1: An RNAi screen of microtubule-regulatory proteins identifies MARK2/Par1 as an effector of Rac1-mediated microtubule growth. Yukako Nishimura, Kathryn Applegate, Gaudenz Danuser, Clare Waterman Proper regulation of microtubule (MT) assembly dynamics is essential for directed cell migration. Microtubule dynamics in migrating cells are spatially regulated by Rho GTPases. We have previously shown that activated Rac1 induces MT net growth by suppressing catastrophe and increasing growth velocity, and that Rac1 activity is required for polarized MT growth in the leading edge of migrating cells. We identified a necessary, but not sufficient PAK kinase-mediated pathway downstream of Rac1 that promoted MT growth. Therefore, we hypothesized that additional factors promote MT net growth downstream of Rac1. To find these factors, we performed a RNAi screen in human U2OS osteosarcoma cells to determine if known MT-regulatory proteins were required for constitutively activated Rac1 promotion of MT growth. To analyze MT dynamics, we imaged fluorescent-tagged EB3, a MT plus-end binding protein that serves as a probe for the position of MT ends, and tracked the motion of EB3 comets in time-lapse movies using an automated computer program. Our results indicate that depletions of several MT-binding proteins change the growth rate of MT in activated Rac1-expressing cells. We have focused on MARK2, a microtubule affinity-regulating kinase homologous to the C. elegans polarity protein Par1, whose depletion reduces the number of elongated MTs in the leading edge of Rac1-activated cells. We are currently testing how MARK2 is involved in promoting MT growth downstream of Rac1 and it requirement in cell migration. Amanuscript describing these results is being prepared for publication. Project 2: MCAK Activity Controls Interphase Microtubule Dynamics and Directed Cell Migration. Myers, K.A.;Applegate, K;Danuser G.;and Waterman, C.M. Directional cell migration is initiated through extracellular stimuli that coordinate changes in the cytoskeleton to establish a polarized cellular morphology. Cell polarity can be achieved through regional regulation of microtubule (MT) dynamics, including MT growth toward the leading edge and MT shortening in the cell rear. Mitotic Centromere Associated Kinesin (MCAK) is a MT depolymerase that is down-regulated in mitosis by Aurora kinase phosphorylation. While its mitotic functions have been well-characterized, whether MCAK regulates MT dynamics during cell migration is not known. We hypothesize that MCAK is down-regulated locally via a Rac1/Pak1/Aurora-A kinase signaling pathway to establish preferential MT growth toward the leading edge and to promote MT shortening within the cell rear. To test this hypothesis, we performed time-lapse imaging of fluorescently tagged EB3 as a marker of MT plus end growth in HUVEC cells and analyzed MT dynamics and cell behavior under different manipulations of the proposed signaling cascade. We find that MCAK knockdown (KD) produces expected effects on the MT cytoskeleton, including increased levels of tubulin polymer and decreased MT catastrophe frequency. MCAK-KD cells show a reduction in MT polymerization speeds and exhibit a mal-oriented MT array, as well as a statistically significant reduction in cell migration velocity, directional persistence, and distance to origin, indicating a defect in cell migration and/or polarization. These effects are rescued through expression of exogenous wild-type-MCAK, but not by expression of either an inactive (ATPase-dead) MCAK mutant or an MCAK mutant that is incapable of phospho-regulation by Aurora-A kinase. Immunolabeling of cells expressing either constitutively active-Rac1 or constitutively active-Pak1 suggests that Rac1 and Pak1 activities correlate with increased Aurora-A activity, as assayed with a phospho-specific antibody, and also correlate with decreased levels of MCAK expression. These data suggest that interphase regulation of MCAK is achieved downstream of a Rac1/Pak1/Aurora-A signaling pathway in order to locally coordinate MCAK-mediated MT depolymerization as a method to ensure proper cell polarization and motility. Project 3: Regulation of microtubules in migrating endothelial cells in 3D ECMs Ken Myers, Kathryn Applegate, Gaudenz Danuser, Robert Fischer, Clare Waterman Cytoskeletal dynamics driving endothelial cell (EC) branching morphogenesis during angiogenesis are thought to be regulated in part by cellular signals elicited in response to compliance and topology of the extracellular matrix (ECM) via a process termed mechanosensing. We hypothesized that ECM mechanosensing of compliance or topology (2 dimensional vs 3 dimensional ECMs, i.e. ECM dimensionality) could elicit different responses of the microtubule (MT) cytoskeleton to mediate EC branching morphogenesis. To test this, we used novel MT end-tracking software to analyze spatial variations in MT dynamics in ECs in 2D and 3D compliance-controlled ECMs during branching morphogenesis. Pharmacological inhibition showed that MT dynamics negatively regulate EC branching, independent of ECM compliance or dimensionality. Analyzing MT dynamics incompliant 2D and 3D ECMs with or without myosinII inhibition indicated that myosinII down-regulation by compliance mechanosensing promotes fast MT assembly, but we found dimensionality-specific effects on MT growth persistence. Comparing MT dynamics in cell bodies versus cell branches in in 2D and 3D ECMs of varying stiffness revealed faster, more dynamically unstable MT growth in EC bodies and slower, more persistent MT growth in EC branches. Thus, distinct compliance and dimensionality ECM mechanosensing pathways regionally regulate MT dynamics in ECs to guide branching morphogenesis in physically complex ECMs. A manuscript describing these results was submitted to JCB in june, and is currently being revised for resubmission.